The incidence and mortality rates of melanoma have been increasing over the last few decades (Balch et al., J. Clin. Oncol. 27(36): 6199-6206 (Dec. 20, 2009)). The American Cancer Society (ACS) estimates that the lifetime risk of developing melanoma is approximately 1 in 50 for Caucasians, 1 in 1,000 for African-Americans, and 1 in 200 for Hispanics. Overall, melanoma is the sixth most common cancer in men and the seventh most common cancer in women (American Cancer Society (ACS), Melanoma, cancer.org/docroot/CRI/content/CRI_2 4_1x hd What_are the key_statistics_for_melan oma_50.asp?sitearea=(2009)). In 2009, 68,720 new cases of invasive melanoma, and 8,650deaths were reported in the United States (ACS (2009), supra).
Currently, histology is recognized as the gold standard for the diagnosis of melanoma, and it is, therefore, the current gold standard for predicting clinical behavior. Histology is essentially a surrogate marker for predicting clinical outcome. However, histology is the method of choice by default, and it has not been completely validated. Numerous epidemiologic and clinical studies illustrate the limitations of histology. For example, it is well-recognized that a large percentage of histologically malignant-appearing lesions behave in a benign manner; such lesions are referred to as “indolent melanoma.” By contrast, a small percentage of melanomas, which invade the skin to a shallow Breslow's depth, behave in a very malignant and aggressive manner, resulting in metastasis and death.
Sentinel lymph node biopsy is performed in melanoma patients with a high risk for metastases to evaluate the lymph node for metastatic involvement by melanoma. Generally, patients with a melanoma of Breslow's depth greater than about 0.75 mm are biopsied. Such patients have a poor prognosis based on histological factors such as high mitotic rate, ulceration, or Clark's level IV or V. When the sentinel lymph node is involved in melanoma, there is an 80-90% chance that the patient will develop a metastatic disease. Hence, the sentinel lymph node status is the strongest prognostic indicator of melanoma. Those patients in whom the sentinel lymph node is not involved in melanoma are considered to be a cohort of patients with significantly better prognosis compared to those patients in whom the sentinel lymph node is involved in melanoma.
Although the number of melanoma cancer-related deaths continues to increase and the treatment of advanced melanoma continues to show dismal results, there have been several breakthroughs in the past decade. These breakthroughs include the stratification of melanoma into molecular subtypes, which correlate to prognosis (Viros et al., PLoS Med 5(6): e120 (Jun. 3, 2008)), as well as targeted therapy, which can be tailored to the specific activated oncogenic pathway (Hodi et al., J. Clin. Oncol. 26(12): 2046-2051 (Apr. 20, 2008); and Jiang et al., Clin. Cancer Res. 14(23): 7726-7732 (Dec. 1, 2008)). For example, specific targeted inhibitors, such as BRAF inhibitors or CKIT inhibitors, such as imatinib mesylate, have been successfully used to treat patients with advanced melanoma (Hodi et al. (2008), supra; and Lutzky et al., Pigment Cell Melanoma Res. 21(4): 492-493 (August 2008)). Hence, identifying specific oncogenic pathways in melanoma can help stratify melanoma patients prognostically and can help predict therapeutic results.
In addition to somatic mutations, copy number aberrations through gain of specific oncogenes or loss of specific tumor suppressor genes are highly characteristic of melanoma. The genomic classification of malignant melanoma based on patterns of gene copy number alterations has been proposed; such classification reportedly would enable rational patient selection for treatment (U.S. Pat. App. Pub. No. 2010/0145897 and Int'l Pat. App. Pub. No. WO 2010/051319). Comparative genomic hybridization (CGH) studies show that 95% of melanomas have chromosomal copy number aberrations (Bastian et al., Cancer Res. 58(10): 2170-2175 (May 15, 1998)). Frequent chromosomal copy number losses include deletions at 9p (82%), 10q (63%), 6q (28%), and 8p (22%). Frequent copy number gains may occur at 7q (50%), 8q (34%), 6p (28%), and 1q (25%), among others (Bastian et al. (1998), supra). Melanomas on acral sites reportedly have significantly more aberrations involving chromosomes 5p, 11q, 12q, and 15, as well as focused gene amplifications (Bastian et al., Amer. J. Path. 163: 1765-1770 (2003)). An algorithm using signal counts from a combination of four fluorescent in situ hybridization (FISH) probes targeting chromosome 6p25, 6 centromere, 6q23, and 11q13 provides the highest diagnostic discrimination between melanomas and nevi with 86.7% sensitivity and 95.4% specificity (Gerami et al., Am. J. Surgical Path. 33: 1146-1156 (2009)); see, also, Gerami et al., Arch. Dermatol. 144(9): 1235-1236 (September 2008), and Pouryazdanparast et al., Amer. J. Dermatopathol. 31(4): 402-403 (June 2009)). Melanomas with wild-type BRAF or N-RAS reportedly have frequent increases in the number of copies of the genes for cyclin-dependent kinase 4 (CDK4) and cyclin D1 (CCND1) (Curtin et al., New England J. of Med. 353: 2135-2147 (Nov. 17, 2005)). A small subset of Spitz nevi reportedly shows an isolated gain of the short arm of chromosome 11p, which has not been observed in melanomas (Bastian, Recent Results Cancer Res. 160: 92-99 (2002); and Bastian et al. (2003), supra). However, while copy number gains have been linked to diagnosis of melanoma, to date there has been no linkage to prognosis. The ability to relate genetic alterations to prognosis of melanoma would help to improve prognostication and management of patients with conventional therapies and could help identification of therapeutic targets.
Copy number gains of specific oncogenes have been linked to prognosis in a number of cancers. For example, amplification of Her-2/neu has been associated with poor prognosis in breast cancers (Tovey et al., Br. J. Cancer 100(5): 680-683 (Mar. 10, 2009)), while elevated copy numbers of the epidermal growth factor receptor (EGFR) gene are highly associated with likely response and survival benefit of non-small cell lung cancer treated with EGFR tyrosine kinase inhibitors (Dahabreh et al., Clin. Cancer Res. 16(1): 291-303 (Jan. 1, 2010)).
The present disclosure seeks to provide a method for prognosing malignant melanoma, including atypical Spitz tumors, in a patient. This and other objects and advantages, as well as additional features, will become apparent from the detailed description provided herein.
In addition to the foregoing, the present disclosure seeks to provide a method for diagnosing malignant melanoma in a patient. It is well-accepted among pathologists that there are several subsets of melanocytic tumors that can be difficult to classify clearly as either benign or malignant (Barnhill et al., Hum. Pathol. 30(5): 513-520 (1999); Corona et al., J. Clin. Oncol. 14(4): 1218-1223 (1996); and McGinnis et al., Arch. Dermatol. 138(5): 617-621 (2002)). In many cases the differential diagnosis includes an entirely benign lesion, such as a spitz nevus, as opposed to a highly lethal, malignant lesion, such as melanoma with spitzoid morphological features. Given the development of new biological therapies for melanoma, it is desirable to be able to distinguish malignant and benign lesions. Added advantages of differential diagnosis include the avoidance of undue psychological burden resulting from an incorrect diagnosis, the determination of appropriate surgical management with or without sentinel lymph node biopsy, and the determination of appropriate systemic therapy.
In addition to conventional microscopy, molecular diagnostic techniques are emerging and are showing promise as diagnostic aids. A four-probe fluorescence in situ hybridization (FISH) assay targeting 6p25 (RREB1), 6q23 (MYB), Cep6 (centromere 6), and 11q13 (CCND1) has been shown to distinguish hisologically unequivocal melanomas from benign nevi with a sensitivity of 86.7% and a specificity of 95.4% (Gerami et al., Am. J. Surg. Pathol. 33(8):1146-56 (2009)). When this probe set and the predetermined criteria were applied to a set of ambiguous melanocytic neoplasms with known follow-up, there was a significant difference in the results among the group with metastasis (6/6 positive) versus the group with no metastasis (6/21 positive). The p-value showing the difference in likelihood of a positive result in the metastasis versus the non-metastasis group was highly significant, i.e., less than 0.003 by Fisher's exact test. The foregoing probe set was also helpful in distinguishing nevoid melanoma from mitotically active nevi (Gerami et al., Am. J. Surg. Pathol. 33(12): 1783-1788 (2009)), blue nevus-like metastasis from epithelioid blue nevus (Pouryazdanparast et al., Am. J. Surg. Pathol. 33(9): 1396-400 (2009)), and conjunctival melanoma from conjunctival nevi (Busam et al., J. Cutan. Pathol. 37(2): 196-203 (2010)). However, a separate analysis of a series of spitzoid melanomas showed a sensitivity of 70% in this subset of lesions (Gammon et al., Am. J. Surg. Pathol., “Enhanced Detection of Spitzoid Melanomas Using Fluorescence In Situ Hybridization With 9p21 as an Adjunctive Probe” (Epub ahead of print, Oct. 10, 2011)). Hence, while the development of the above-described probe set was a significant advancement in molecular diagnosis of melanocytic neoplasms, there is room for improvement in the sensitivity of the assay, particularly for spitzoid melanomas. While the above-described probe set is relatively highly specific, the presence of tetraploid cells in spitzoid neoplasms can occasionally cause difficulty in interpreting the results of the FISH (Isaac et al., Am. J. Dermatopathol. 32(2): 144-148 (2010)). Other objects and advantages of the method for diagnosing malignant melanoma in a patient, as well as additional features, will become apparent from the detailed description provided herein.